Dasabuvir alleviates 5-fluorouracil-induced intestinal injury through anti-senescence and anti-inflammatory

5-Fluorouracil (5-Fu) is a basic drug that is used to treat colorectal cancer. Patients who receive 5-Fu chemotherapy often experience side effects that affect the digestive system, such as intestinal injury and diarrhoea, which significantly affect patient compliance with anticancer treatment and quality of life. Therefore, identifying approaches to treat or prevent these side effects is urgent. Dasabuvir (DSV) is a hepatitis C virus inhibitor, but its impact on 5-Fu-induced intestinal injury remains unknown. Our study investigated the effects of DSV on 5-Fu-induced intestinal injury in HUVECs, HIECs and male BALB/c mice. We found that 5-Fu caused intestinal damage by inducing senescence, increasing inflammatory factor expression, and generating oxidative stress. Compared with 5-Fu treatment alone, DSV inhibited senescence by reducing senescence-β-galactosidase (SA-β-gal) activity, the senescence-associated secretory phenotype (SASP, including IL-1, IL-6, and TNF-α) and senescence marker expression levels (p16, p21, and p53). Moreover, the anti-senescence effect of DSV was achieved by inhibiting the mTOR signaling pathway. DSV increased antioxidant enzyme levels and alleviated intestinal tissue injury in mice. In addition, DSV suppressed the 5-Fu-induced increase the diarrhoea scores and ameliorated the weight loss, food intake and water intake of the mice. Overall, this study indicated that DSV could be used to treat chemotherapy-induced intestinal damage.


DSV alleviates 5-Fu-induced endothelial cell and intestinal epithelial cell senescence
HUVECs are often used as a model to induce premature cellular senescence.Our published article reported that 5-Fu induced senescence in the highest percentage of cells at a concentration of 1 μM 21,22 .Therefore, 1 μM 5-Fu was used to establish a senescence model in HUVECs and HIECs.To study the effect of DSV on 5-Fu-induced senescence, HUVECs were incubated with different concentrations of DSV combined with 1 μM 5-Fu for 6 days.Senescence was assessed by determining the percentage of SA-β-gal-positive cells.While 5-Fu increased the percentage of SA-β-gal-positive cells, 3 to 7 µM DSV notably reduced the percentage of SA-β-gal-positive cells, indicating that DSV can alleviate endothelial cell senescence (Fig. 1a-c).We hypothesized that the intestinal side effects in patients who receive chemotherapy are related to the senescence-inducing effects of the chemotherapeutic agents.To test this hypothesis, we performed preliminary experiments using HIECs.5-Fu induced the senescence of HIECs, while DSV reversed this increase in the proportion of SA-β-gal-positive cells (Fig. 1d, e).Compared with those in the control group, cells that were treated with DSV (5 µM or higher) exhibited a flattened morphology and less distinct cell outlines.However, after treatment with 3 µM DSV, the cell morphology appeared normal, with a low percentage of ageing cells.In addition, treatment with DSV alone did not induce HUVEC or HIEC senescence.Subsequently, we investigated the impact of coculture with drugs on cell viability.As shown in Fig. 1f, g, our findings revealed that there was no statistically significant effect on cell viability When DSV was used at concentrations ≤ 3 µM, whether DSV given alone or in combination with 5-Fu.DSV (3 μM) was selected for subsequent experiments.To further investigate the anti-senescence effect of DSV in HIECs, we examined the protein and gene expression of the senescence-related markers p16, p21 and p53.5-Fu upregulated p16 protein expression and p53 and p21 mRNA expression in HIECs.Cotreatment with DSV downregulated the p16 protein expression and the p53 and p21 mRNA expression.Moreover, DSV alone did not affect these indicators (Fig. 1h-k).Hence, these results indicated that DSV alleviates 5-Fu-induced endothelial cell and intestinal epithelial cell senescence.

DSV attenuates intestinal senescence in 5-Fu-treated mice
Next, we further investigated the anti-senescence effects of DSV by establishing a diarrhoea mouse model.During treatment, several clinical parameters and signs were recorded.Compared with those in the control group, the mice that were treated with 5-Fu lost significantly more weight, while DSV prevented weight loss.(Fig. 2a) (body weight of each group before sacrifice: control group: 30.88 g ± 1.043, 5-Fu group: 23.30 g ± 0.952, DSV group:26.20g ± 0.671).Starting on the second day, the mice in the 5-Fu group began to develop diarrhoea.From Days 6 to 10, the diarrhoea score of the 5-Fu group decreased in response to DSV treatment (Fig. 2b).DSV ameliorated the 5-Fu-induced decrease in food intake and water intake (Fig. 2c, d).To observe the antisenescence effect of DSV, frozen intestinal tissue sections were subjected to SA-β-gal staining.As indicated in the figures, three kinds of intestinal tissue sections from the 5-Fu group showed varying degrees of staining, and tissue damage could be observed.Compared to the control, 5-Fu resulted in deep staining of ileum and colonic villi and light staining of rectal villi.DSV reduced SA-β-gal staining in intestinal villi (Fig. 2e-g).Moreover, we evaluated the protein expression of p16 and gene expression of p21 and p53 in colon tissues.The protein expression of p16 was upregulated by 5-Fu and downregulated by DSV (Fig. 2h, i).The mRNA levels of p53 and p21 were increased in the 5-Fu group and decreased in the DSV group (Fig. 2j, k).These results showed that DSV attenuates intestinal senescence in 5-Fu-treated mice.

DSV inhibits 5-Fu-induced senescence by inhibiting mTOR
We next investigated the mechanism by which DSV inhibits 5-Fu-induced senescence.mTOR is a pivotal regulatory molecule that is involved in regulating senescence, and inhibition of mTOR can delay senescence.As shown in the figures, mTOR protein levels in HIECs were elevated by 5-Fu treatment on Days 1 and 3 and then downregulated by DSV treatment (Fig. 3a, b).Next, we added mTOR activator MHY1485 to HIECs based on

DSV inhibits 5-Fu-induced abnormal levels of inflammatory factors and antioxidant enzymes in HIECs
Inflammatory cytokines, such as IL-1, IL-6, and TNF-α, play key roles in the initiation and maintenance of cell senescence.Decreases in the levels of antioxidant enzymes such as Catalase (CAT) and Superoxide Dismutase www.nature.com/scientificreports/(SOD) induce senescence 24 .We measured changes in the expression levels of these indicators in HIECs.After HIECs were treated with 5-Fu, the IL-1, IL-6, and TNF-α levels were increased on Day 3, and the SOD and CAT levels decreased on Day 1.However, DSV prevented these changes (Fig. 4a-e).To further investigate the antiinflammatory effects of DSV, HIECs were treated with 1 μM 5-Fu and 3 μM DSV for 1 day and 3 days.The p-p65 and p-p38 protein levels were upregulated by 5-Fu but downregulated by DSV (Fig. 4f-h).

DSV inhibits 5-Fu-induced intestinal injury and abnormal levels of inflammatory factors and antioxidant enzymes in mice
We next examined the changes in the indicators described above in colon tissue.5-Fu increased the levels of IL-1, IL-6, and TNF-α and decreased the levels of SOD and CAT.Treatment with DSV prevented these changes (Fig. 5a-e).5-Fu treatment increased the p-p65 and p-p38 protein levels in mouse colon tissues, whereas DSV treatment prevented these changes (Fig. 5f-h).To observe the changes in intestinal morphology, HE staining was performed on colon, ileum and rectum samples from each group.The intestinal tissues of the control group had a normal intestinal structure, with intact villi, mucosal submucosa and basal layer.5-Fu treatment destroyed the intestinal villi and structures, while DSV ameliorated this damage (Fig. 5i).These results demonstrated that DSV alleviates 5-Fu-induced oxidative stress, inflammation and intestinal injury induced by the above factors.

DSV increases 5-Fu cytotoxicity in HCT116 cells
To investigate the effect of DSV on the antitumour effects of 5-Fu, we examined the synergistic effect of DSV on the anticancer properties of 5-Fu.We examined the effects of 5-Fu and DSV, alone and combined, on cell viability in HCT116s.The findings indicated that 5-Fu and DSV reduce HCT116 cell viability in a concentration-dependently, respectively.The coadministration of 5-Fu and DSV had a more pronounced cytotoxic effect on cancer cells than single-drug treatments (Fig. 6a).Subsequent experiments involved treating HCT116 cells with 3 µM 5-Fu in combination with 3 µM DSV to assess the effects on colony formation.Furthermore, colony formation assays revealed significant inhibition of cancer cell colony formation by the combined treatment (Fig. 6b, c), suggesting that the combination of 5-Fu and DSV could effectively suppress the proliferation of colorectal cancer cells.

Discussion
An increasing number of studies have suggested that chemotherapy and radiotherapy can induce cell senescence, which leads to side effects and affects therapeutic efficacy 25 .In this study, we demonstrated that 5-Fu induced the senescence of HUVECs, HIECs and mouse intestinal tissues.Moreover, 5-Fu increased the levels of senescence-related markers.DSV is a nonstructural (NS) 5B nonnucleoside hepatitis C virus inhibitor that is used in combination with other direct antiviral agents for the treatment of chronic hepatitis C virus infection, and it has a high cure rate and few side effects 26 .Interestingly, we found that DSV alleviated 5-Fu-induced senescence in endothelial cells, intestinal epithelial cells and mouse intestinal tissues via inhibition of the mTOR signalling pathway.Moreover, DSV alleviated 5-Fu-induced inflammation and oxidative stress both in vitro and in vivo.
Intestinal mucositis induced by 5-Fu in mice results in clinical manifestations including diarrhoea, weight loss, and intestinal tissue damage 27 , which were also observed in this study; these changes were ameliorated by DSV treatment.In conclusion, DSV is expected to be used as a drug for the treatment of 5-Fu-induced intestinal injury.According to the results, 5-Fu activated mTOR in intestinal epithelial cells and mouse colon tissues.mTOR signalling contributes to the regulation of cellular senescence 28 .As an mTOR inhibitor, rapamycin slows the process of senescence and the occurrence and progression of age-related diseases in mammals 29,30 .Inhibiting mTOR affects the expression levels of inflammatory factors.Rapamycin suppresses the secretion of TNF-α, IL-1 and IL-3 31 .mTOR inhibitors have therapeutic effects on inflammatory bowel disease 32 .mTOR activation was observed in the colon of Crohn's disease patients 33 .Overactivation of mTOR leads to the apoptosis of mouse intestinal epithelial cells and induces colitis 34 .Inhibition of p-mTOR expression can treat ulcerative colitis and reduce intestinal damage.We demonstrated that DSV exerts an anti-senescence effect by inhibiting mTOR signalling, which alleviates intestinal damage.
NF-κB, which consists of several transcription factors (p52, p65, p17, c-REL and ReIB), participates in the regulation of homeostasis, inflammation and immunity 35,36 .Activation of p65 has been observed in colitis, and inhibition of NF-κB may treat inflammatory bowel disease 37 .Inhibition of NF-κB activity can reduce the 5-Fu-induced increase in the IL-1β and TNF-α levels and reduce intestinal injury 38 .The p65 pathway is also involved in the regulation of cellular senescence.Studies have shown that p65 activation is a key pathway for MSC senescence and increased inflammatory cytokine expression 39 .NF-κB is a strong signal that triggers the secretion of SASP components 40 .Senescent cells secrete many inflammatory cytokines through the p38MAPK and p65 pathways 41 .Our results demonstrated that DSV suppressed the 5-Fu-induced increase in the levels of SASP proteins (IL-1β, IL-6, and TNF-α), p-p65, and p38MAPK.
The pathogenesis of mucositis is complex and involves many processes.In addition to inflammatory processes, oxidative stress also causes apoptosis and tissue damage.Reactive oxygen species directly induce tissue damage and inflammatory processes 10,42 .Oxidative stress is also a characteristic of senescence, manifesting as increased levels of oxidative factors and decreased levels of antioxidant enzymes 24 .In our previous study, we demonstrated that 5-Fu led to an increase in ROS levels 21 .Enhancing the activity of antioxidant enzymes can alleviate oxidative stress damage 43 .The expression levels of two antioxidant enzymes (SOD and CAT) were decreased by 5-Fu but increased by DSV.
The senescence-inducing effect of chemotherapeutic drugs during cancer treatment has gradually attracted increasing attention.Inflammatory factors that are secreted by senescent cells participate in the maintenance of senescence, gradually leading to chronic inflammation.Chronic inflammation not only reduces the efficacy of chemotherapy but also causes side effects, which impose health and economic burdens on patients 25 .Some studies have shown that cells with TIS and SASP factors that are secreted by cells with TIS lead to lung inflammation, bone marrow suppression and heart failure in mice, and the removal of senescent cells can delay the progression of adverse reactions 44,45 .Our present study showed that inhibiting 5-Fu-induced intestinal senescence alleviated intestinal damage and diarrhoea.Moreover, DSV can enhance the cytotoxicity of 5-FU to HCT116 and enhance the anticancer effect.
We studied the efficacy of DSV in treating 5-Fu-induced intestinal mucositis.In conclusion, DSV alleviated 5-Fu-induced intestinal mucositis by inhibiting senescence, inhibiting inflammation, regulating oxidative stress and relieving clinical symptoms.These findings suggest that DSV can be used as an anti-senescence drug to treat 5-Fu-induced intestinal mucositis.

Induction of intestinal mucositis and treatment protocol
The animal experiments that were conducted for this study were carried out in accordance with the ARRIVE guidelines, and the details are as follows.Eighteen mice were randomly divided equally into three groups using the rand () function of Excel (Microsoft).The control group received phosphate-buffered saline (PBS, B548117, Sangon Biotech, Shanghai); the 5-Fu group received 40 mg/kg 5-Fu (H12020959, Tianjin Jinyao Pharmaceutical Co.) through intraperitoneal injection; and the DSV group received 5-Fu similar to the 5-Fu group and received 10 mg/kg DSV (S7650, Selleck, Shanghai, China).The mice in the three groups were treated by intraperitoneal injection.5-Fu was given once every two days (on Days 2, 4, 6, 8, and 10), while DSV was given once daily.The dosage of 5-Fu that was used in this study was based on our published article 21 .The dosage of DSV was selected according to a published article and our preliminary experiment 46 .For each animal, three different investigators were involved: two investigators (S.H. and J.X.) administered the treatment based on the randomization table, and a third investigator (D.Q.) recorded changes in body weight, food intake and water intake at the same time every day.The stool of the mice was evaluated according to the following criteria, and the diarrhoea score was recorded by an investigator who was blinded to the treatments.The diarrhoea score was used to assess the severity of diarrhoea based on the following criteria: 0: normal stool; 1: squishy stool (mild diarrhoea); 2: moist and unshaped stool (moderate diarrhoea); and 3: liquid stool (severe diarrhoea) 43 .After the experiment, the mice were sacrificed by cervical dislocation of the neck, and the colon, ileum, and rectal tissues were collected from each mouse.The intestinal tissues of three mice in each group were used to generate frozen sections and paraffin sections, and the intestinal tissues of the other three mice were used for immunoblotting and RT-qPCR experiments.Therefore, each intestinal tissue sample was considered an experimental unit.No exclusion or inclusion criteria were applied to the mice that were used in this study.

Cell drug treatments
Dasabuvir was first dissolved in dimethyl sulfoxide (DMSO) at 1000 mM (stock concentration) and then diluted with culture medium to the appropriate concentration.The final concentration of DMSO was less than 0.1%.

Cell viability assay
HUVECs, HIECs, and HCT116 cells were separately cultured in 96-well plates (1000 cells per well, 3 replicate wells, 24 h), and each group was cultured with the indicated drugs for 72 h.After incubation, the medium was removed, and a mixture of CCK-8 and medium (1:9 volume ratio) was added to each well at 100 μL/well and incubated for 1 h.A microplate absorbance reader (Bio-Rad, USA) was used to measure the optical density (OD) at 450 nm.The concentrations of the above reagents are indicated in the corresponding figure legends.

Colony-formation efficiency assay
HCT116 cells were seeded in six-well plates at a density of 2000 cells per well and cultured with the indicated concentrations of DSV, 5-Fu or the combination for 72 h.After incubation, the cells were cultured in drug-free medium for 10 days.After colonies formed, the cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 15 min.The colonies were counted and analyzed using ImageJ 1.52i (NIHR, USA).

SA-β-Gal staining
After the mice were sacrificed, intestinal tissues were fixed in OCT at low temperature and sliced into 4-μm sections.The SA-beta-gal activity of HUVECs, HIECs and intestinal samples was measured using a senescenceassociated-β-galactosidase (SA-β-Gal) staining kit according to the manufacturer's instructions.The senescent cells and sections were identified by blue/green staining under an optical microscope, and images were captured.The percentage of SA-β-Gal-positive cells was determined by counting 1,000 cells in 7 random fields for each group.

Histological staining
Intestinal tissues were fixed in 4% paraformaldehyde and embedded in paraffin blocks.Paraffin-embedded tissue sections (5 µm thick) were deparaffinized and stained using a haematoxylin-eosin (HE) staining kit (Solarbio, Beijing, China).The samples were viewed and images were acquired using a light microscope.